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CRYP-2/cPTPRO is a neurite inhibitory repulsive guidance cue for retinal neurons in vitro.

Stepanek L, Sun QL, Wang J, Wang C, Bixby JL - J. Cell Biol. (2001)

Bottom Line: We found that the extracellular domain of cPTPRO is an antiadhesive, neurite inhibitory molecule for retinal neurons.This chemorepulsive effect could be regulated by the level of cGMP in the growth cone.Immunohistochemical examination of the retina indicated that cPTPRO has at least one heterophilic binding partner in the retina.

View Article: PubMed Central - PubMed

Affiliation: Neuroscience Program, University of Miami School of Medicine, Miami, FL 33136, USA.

ABSTRACT
Receptor protein tyrosine phosphatases (RPTPs) are implicated as regulators of axon growth and guidance. Genetic deletions in the fly have shown that type III RPTPs are important in axon pathfinding, but nothing is known about their function on a cellular level. Previous experiments in our lab have identified a type III RPTP, CRYP-2/cPTPRO, specifically expressed during the period of axon outgrowth in the chick brain; cPTPRO is expressed in the axons and growth cones of retinal and tectal projection neurons. We constructed a fusion protein containing the extracellular domain of cPTPRO fused to the Fc portion of mouse immunoglobulin G-1, and used it to perform in vitro functional assays. We found that the extracellular domain of cPTPRO is an antiadhesive, neurite inhibitory molecule for retinal neurons. In addition, cPTPRO had potent growth cone collapsing activity in vitro, and locally applied gradients of cPTPRO repelled growing retinal axons. This chemorepulsive effect could be regulated by the level of cGMP in the growth cone. Immunohistochemical examination of the retina indicated that cPTPRO has at least one heterophilic binding partner in the retina. Taken together, our results indicate that cPTPRO may act as a guidance cue for retinal ganglion cells during vertebrate development.

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cPTPRO inhibits LN-induced neurite outgrowth. Dissociated E6 chick retinal neurons were allowed to grow overnight, fixed, and photographed through a phase–contrast microscope. (A) PDL alone, (B) PDL plus10 μg/ml LN, (C) PDL plus LN/50 μg/ml cPTPRO–Fc, or (D) PDL plus LN/100 μg/ml cPTPRO–Fc. Neurons grew long neurites on PDL–LN. Neurites were noticeably fewer and shorter when cPTPRO was mixed with LN, especially at the higher concentration (D). Bar, 100 μm.
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fig5: cPTPRO inhibits LN-induced neurite outgrowth. Dissociated E6 chick retinal neurons were allowed to grow overnight, fixed, and photographed through a phase–contrast microscope. (A) PDL alone, (B) PDL plus10 μg/ml LN, (C) PDL plus LN/50 μg/ml cPTPRO–Fc, or (D) PDL plus LN/100 μg/ml cPTPRO–Fc. Neurons grew long neurites on PDL–LN. Neurites were noticeably fewer and shorter when cPTPRO was mixed with LN, especially at the higher concentration (D). Bar, 100 μm.

Mentions: Although nothing is known concerning the activities of type III RPTPs, the ECDs of several type II RPTPs have been shown to promote neurite growth in vitro (Burden-Gulley and Brady-Kalnay, 1999; Drosopoulos et al., 1999; Ledig et al., 1999a; Wang and Bixby, 1999). Furthermore, cPTPRO is localized on RGC axons and growth cones, putting it in the right location to influence axon growth (Ledig et al., 1999b). To test whether the ECD of cPTPRO can regulate neurite growth from retinal neurons, we compared the growth of retinal neurites on substrates of LN with growth on LN mixed with cPTPRO–Fc. As cPTPRO is antiadhesive, we first coated substrates with PDL to allow LN-independent adhesion of retinal neurons. Retinal neurons adhered well, but did not grow neurites when cultured overnight on PDL alone (Fig. 5 A). Neurons adhered and grew numerous processes on an LN–PDL substrate (Fig. 5 B). The presence of PDL allowed strong neuronal adhesion when cultures were grown on a cPTPRO–Fc substrate, but neurons mainly failed to extend neurites, showing that cPTPRO does not promote retinal neurite growth (unpublished data). More interestingly, LN-induced neurite formation was substantially inhibited by the presence of cPTPRO (Fig. 5, C and D). Quantification of neurite outgrowth confirmed that the presence of cPTPRO inhibited, in a dose-dependent manner, both the percentage of cells with neurites and the average length of individual neurites (see Fig. 7, A and B) . In control experiments, mixing LN with IgG did not affect either parameter of neurite growth, even when the IgG was present at higher concentrations than the cPTPRO–Fc (see Fig. 7). To ensure that the inhibitory effect of cPTPRO was not due to competition for binding to the culture substrate, we performed experiments in which LN was spotted onto the substrate before coating with cPTPRO. In two experiments, neurite outgrowth (measured as percentage of neurons with neurites) was inhibited 61% by PTPRO added to the substrate after the LN coating, compared with 69% inhibition by PTPRO added simultaneously with the LN. Taken together, these data indicate that the cPTPRO ECD can inhibit retinal neurite growth induced by substrate-bound LN.


CRYP-2/cPTPRO is a neurite inhibitory repulsive guidance cue for retinal neurons in vitro.

Stepanek L, Sun QL, Wang J, Wang C, Bixby JL - J. Cell Biol. (2001)

cPTPRO inhibits LN-induced neurite outgrowth. Dissociated E6 chick retinal neurons were allowed to grow overnight, fixed, and photographed through a phase–contrast microscope. (A) PDL alone, (B) PDL plus10 μg/ml LN, (C) PDL plus LN/50 μg/ml cPTPRO–Fc, or (D) PDL plus LN/100 μg/ml cPTPRO–Fc. Neurons grew long neurites on PDL–LN. Neurites were noticeably fewer and shorter when cPTPRO was mixed with LN, especially at the higher concentration (D). Bar, 100 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196468&req=5

fig5: cPTPRO inhibits LN-induced neurite outgrowth. Dissociated E6 chick retinal neurons were allowed to grow overnight, fixed, and photographed through a phase–contrast microscope. (A) PDL alone, (B) PDL plus10 μg/ml LN, (C) PDL plus LN/50 μg/ml cPTPRO–Fc, or (D) PDL plus LN/100 μg/ml cPTPRO–Fc. Neurons grew long neurites on PDL–LN. Neurites were noticeably fewer and shorter when cPTPRO was mixed with LN, especially at the higher concentration (D). Bar, 100 μm.
Mentions: Although nothing is known concerning the activities of type III RPTPs, the ECDs of several type II RPTPs have been shown to promote neurite growth in vitro (Burden-Gulley and Brady-Kalnay, 1999; Drosopoulos et al., 1999; Ledig et al., 1999a; Wang and Bixby, 1999). Furthermore, cPTPRO is localized on RGC axons and growth cones, putting it in the right location to influence axon growth (Ledig et al., 1999b). To test whether the ECD of cPTPRO can regulate neurite growth from retinal neurons, we compared the growth of retinal neurites on substrates of LN with growth on LN mixed with cPTPRO–Fc. As cPTPRO is antiadhesive, we first coated substrates with PDL to allow LN-independent adhesion of retinal neurons. Retinal neurons adhered well, but did not grow neurites when cultured overnight on PDL alone (Fig. 5 A). Neurons adhered and grew numerous processes on an LN–PDL substrate (Fig. 5 B). The presence of PDL allowed strong neuronal adhesion when cultures were grown on a cPTPRO–Fc substrate, but neurons mainly failed to extend neurites, showing that cPTPRO does not promote retinal neurite growth (unpublished data). More interestingly, LN-induced neurite formation was substantially inhibited by the presence of cPTPRO (Fig. 5, C and D). Quantification of neurite outgrowth confirmed that the presence of cPTPRO inhibited, in a dose-dependent manner, both the percentage of cells with neurites and the average length of individual neurites (see Fig. 7, A and B) . In control experiments, mixing LN with IgG did not affect either parameter of neurite growth, even when the IgG was present at higher concentrations than the cPTPRO–Fc (see Fig. 7). To ensure that the inhibitory effect of cPTPRO was not due to competition for binding to the culture substrate, we performed experiments in which LN was spotted onto the substrate before coating with cPTPRO. In two experiments, neurite outgrowth (measured as percentage of neurons with neurites) was inhibited 61% by PTPRO added to the substrate after the LN coating, compared with 69% inhibition by PTPRO added simultaneously with the LN. Taken together, these data indicate that the cPTPRO ECD can inhibit retinal neurite growth induced by substrate-bound LN.

Bottom Line: We found that the extracellular domain of cPTPRO is an antiadhesive, neurite inhibitory molecule for retinal neurons.This chemorepulsive effect could be regulated by the level of cGMP in the growth cone.Immunohistochemical examination of the retina indicated that cPTPRO has at least one heterophilic binding partner in the retina.

View Article: PubMed Central - PubMed

Affiliation: Neuroscience Program, University of Miami School of Medicine, Miami, FL 33136, USA.

ABSTRACT
Receptor protein tyrosine phosphatases (RPTPs) are implicated as regulators of axon growth and guidance. Genetic deletions in the fly have shown that type III RPTPs are important in axon pathfinding, but nothing is known about their function on a cellular level. Previous experiments in our lab have identified a type III RPTP, CRYP-2/cPTPRO, specifically expressed during the period of axon outgrowth in the chick brain; cPTPRO is expressed in the axons and growth cones of retinal and tectal projection neurons. We constructed a fusion protein containing the extracellular domain of cPTPRO fused to the Fc portion of mouse immunoglobulin G-1, and used it to perform in vitro functional assays. We found that the extracellular domain of cPTPRO is an antiadhesive, neurite inhibitory molecule for retinal neurons. In addition, cPTPRO had potent growth cone collapsing activity in vitro, and locally applied gradients of cPTPRO repelled growing retinal axons. This chemorepulsive effect could be regulated by the level of cGMP in the growth cone. Immunohistochemical examination of the retina indicated that cPTPRO has at least one heterophilic binding partner in the retina. Taken together, our results indicate that cPTPRO may act as a guidance cue for retinal ganglion cells during vertebrate development.

Show MeSH
Related in: MedlinePlus